Persistent Inflammation and Angiogenesis during Wound Healing in K14-Directed Hoxb13 Transgenic Mice  Judith A. Mack, Edward V. Maytin  Journal of Investigative Dermatology  Volume 130, Issue 3, Pages 856-865 (March 2010) DOI: 10.1038/jid.2009.305 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Generation and verification of K14-Hoxb13 transgenic (TG) mice. (a) Schematic of the K14-Hoxb13 transgene construct. Primers 1 and 3 identify the Hoxb13 transgene; Primers 2 and 3 identify both the transgene and the endogenous Hoxb13 gene. (b) Verification of transgene expression by western blot (left) and immunostaining (right) of transiently transfected rat epidermal keratinocytes with anti-Flag and anti-Hoxb13 antibodies; anti-actin, internal loading control. (c) PCR genotyping of selected founder mice; lanes 3 and 4 are positive for the Hoxb13 transgene (upper bands). The lower band indicates both the transgene and endogenous Hoxb13. (d) Western blot of Hoxb13 TG skin extracts with anti-Flag antibody; anti-keratin-14, internal loading control. The lower band (dashed box) represents the Flag-Hoxb13 fusion protein; note its complete absence in the lane from wild-type littermate (WTLM) skin. NS, nonspecific bands. A similar sized NS band was also seen in the western blot shown in panel b. Transgenic strains 1 and 3 (TG1, TG3) express relatively high levels of transgene product compared with strains 2, 4, and 5. (e) Immunostaining of Hoxb13 TG strain 3 (TG3) with anti-Flag antibody. Transgene product is detected in the nuclei of basal keratinocytes of the epidermis and outer root sheath of hair follicles (white arrowheads). Scale bar=100μm. Journal of Investigative Dermatology 2010 130, 856-865DOI: (10.1038/jid.2009.305) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Transgenic (TG) mice expressing high levels of epidermal Hoxb13 exhibit abnormal wound healing compared with low expressors or wild-type littermates (WTLMs). Following general anesthesia and shaving of the dorsal hair, a single 5-mm full-thickness excisional wound was made on the upper back of six pairs of mice (a K14-Hoxb13 TG mouse and its respective WTLM) derived from six different founder strains. The wounds were photographed every other day. (a) Wounds from TG strain 3 (a high Hoxb13 expressor) are shown, along with its WTLM. Note that in the WTLM at day 11 post-wounding, the crust had resolved and the wound was completely closed. By contrast, a large fibrin crust persisted at the wound site in the Hoxb13 TG mouse. (b) At the times indicated, the area of the wound and crust was measured using photography and digital analysis. Data from three strains that weakly expressed the Hoxb13 transgene were pooled (low expressors, left graph, open circle), as were their corresponding WTLM (left graph, closed circle). The strains that strongly expressed the transgene were grouped in a similar manner (high expressors, right graph). Note that for all times up to day 7, all Hoxb13 TG mice showed a larger wound-crust area than the WTLM. This difference persisted beyond day 7 and was statistically significant in the high-expressing Hoxb13 TG mice; *P<0.05, **P<0.01, and ***P<0.0005. Journal of Investigative Dermatology 2010 130, 856-865DOI: (10.1038/jid.2009.305) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Hoxb13 overexpression in the epidermis leads to a highly abnormal wound morphology. Hematoxylin and eosin stains of 11-day-old excisional wounds are illustrated as follows: (a, b), wild-type littermates (WTLMs); (c), a low-expressing Hoxb13 transgenic (TG) mouse; (d, e), high-expressing Hoxb13 TG mice. The founder strains are indicated. (a, b) At 11 days post-wounding, a healthy bed of granulation tissue (1) and a completely intact epidermis (2) with a loosely woven stratum corneum (3) was observed in WTLM wounds. (c) Wounds from a low-expressing TG mouse contained a granulation bed that was healthy but contained increased inflammatory cells (4; area within the dashed region), along with epidermal abnormalities such as increased acanthosis and compact hyperkeratosis (5). (d, e) Wounds from Hoxb13 high-overexpressors remained covered with a dense eschar that contained numerous polymorphonuclear cells (6). Epidermal morphology at those wound sites showed epidermal atrophy overlying the wound bed (7), and irregular epidermal hyperplasia with elongated ridges at the wound edges (8). An increased inflammatory cell infiltrate was observed in the dermal portion of the wound bed in the TG mice (9). Scale bar=100μm. Journal of Investigative Dermatology 2010 130, 856-865DOI: (10.1038/jid.2009.305) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Wounds in Hoxb13 high-expressor mice contain significantly more inflammatory cells than in wild-type mice. Eleven-day-old full-thickness excisional wounds stained with the following antibodies: (a–d) neutrophil-specific, RB6-8C5; (f–i) macrophage-specific, F4/80; (k, l) toluidine blue, for mast cells (arrowheads). Scale bar=100μm. Mouse transgenic (TG) strains 4 and 5 (low expressors) and strains 1 and 3 (high expressors) are illustrated in the panels, while the graphs (e, j, m) show aggregate data for all strains. P-values are indicated for significant differences. NS, not significant. Results represent mean±SEM from at least nine optical fields each from the low Hoxb13 expressors, high Hoxb13 expressors, and wild-type littermates (WTLMs). Journal of Investigative Dermatology 2010 130, 856-865DOI: (10.1038/jid.2009.305) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Blood vessels and lymphatic vessels are enlarged in the skin of K14-Hoxb13 transgenic (TG) mice expressing high levels of the transgene. Staining of frozen sections from 11-day-old full-thickness excisional wounds in K14-Hoxb13 TG3 or matched wild-type littermates, using antisera to the following: (a, b) platelet endothelial cell adhesion molecule-1 (blood vessels); (c, d) lymphatic vessel endothelial receptor-1 (lymphatic vessels). All images are taken from the middle of the wound bed. Dotted lines, epidermal–dermal junction. Arrows, lymphatic vessels. Scale bar=100μm. Journal of Investigative Dermatology 2010 130, 856-865DOI: (10.1038/jid.2009.305) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 VEGF and tumor necrosis factor-α (TNF-α) are differentially upregulated in the skin of Hoxb13 transgenic (TG) mice. (a) Western blot of protein extracts from unwounded skin and from 7-day full-thickness excisional wounds stained with antibodies to VEGF, TNF-α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Locations of molecular weight (MW) markers, in kDa, are shown on the right. The VEGF antibody detects a 43kDa band in unwounded and wounded skin, but the intensity of this band is much greater in the K14-Hoxb13 TG unwounded lanes (asterisks) as compared with the strain-matched wild-type littermates (WT). In wounded skin, high-MW VEGF bands are observed that are significantly more intense in the TG lanes (arrows). The TNF-α antibody detects a 25kDa band that is significantly more intense in the K14-Hoxb13 TG unwounded and wounded lanes. (b) Densiometric analyses of the 43kDa VEGF, 25kDa TNF-α, and GAPDH western blot signals from unwounded skin (n=3 independent K14-Hoxb13 TG mice and three matching WTLM). Journal of Investigative Dermatology 2010 130, 856-865DOI: (10.1038/jid.2009.305) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions